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F Rougeon

Publications and source records attributed to F Rougeon.

At least 19 recordsLinked to original sources
PubMed2002

Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase.

The crystal structure of the catalytic core of murine terminal deoxynucleotidyltransferase (TdT) at 2.35 A resolution reveals a typical DNA polymerase beta-like fold locked in a closed form. In addition, the structures of two different binary complexes, one with an oligonucleotide primer and the other with an incoming ddATP-Co(2+) complex, show that the substrates and the two divalent ions in the catalytic site are positioned in TdT in a manner similar to that described for the human DNA polymerase beta ternary complex, suggesting a common two metal ions mechanism of nucleotidyl transfer in these two proteins. The inability of TdT to accommodate a template strand can be explained by steric hindrance at the catalytic site caused by a long lariat-like loop, which is absent in DNA polymerase beta. However, displacement of this discriminating loop would be sufficient to unmask a number of evolutionarily conserved residues, which could then interact with a template DNA strand. The present structure can be used to model the recently discovered human polymerase mu, with which it shares 43% sequence identity.

Amino Acid Sequence↗
PubMed2001

Chromatin remodeling at the Ig loci prior to V(D)J recombination.

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and kappa L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain J(H) cluster and increased DNase I sensitivity of V(H) and J(H) segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of V(H) segments and a reciprocal increase in the nuclease sensitivity of Vkappa and Jkappa segments. In contrast, J(H) segments remain DNase I sensitive, and their nucleosomal organization is maintained in mu(+) recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and kappa L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.

Acetylation↗
PubMed2001

Terminal deoxynucleotidyl transferase indiscriminately incorporates ribonucleotides and deoxyribonucleotides.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3'-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo- and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo- or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.

Catalysis↗
PubMed2001

The transcriptional response to androgens of the rat VCSA1 gene is amplified by both binary and graded mechanisms.

In higher eukaryotes, gene expression can be highly modified in response to small variations of circulating hormonal inducers. To determine the mechanisms responsible for the 100- to 200-fold enhancement of expression of an androgen-regulated gene, VCSA1, in the acinar cells of rat submandibular glands during puberty, we performed a detailed analysis of VCSA1 expression at the single cell level. Using in situ detection of mature and primary VCSA1 transcripts, we show that VCSA1 expression is activated in only a small proportion of differentiated acinar cells in the presence of low levels of circulating androgens in prepubescent and in castrated males, as well as in females. During the time course of sexual maturation in males, we demonstrate an increase in the proportion of acinar cells expressing VCSA1 and an increase in VCSA1 heterogeneous nuclear RNA and mRNA content in the positive cell population. Finally, we show that changes in the methylation pattern of VCSA1 are correlated with VCSA1 transcriptional activation. These results demonstrate that androgens can, in physiological conditions, elicit both a binary and a graded response. They also provide evidence that the range of gene regulation may be expanded by a transcriptional repression in a majority of cells under basal conditions.

Androgens↗
PubMed2000

Comparison of the two murine terminal [corrected] deoxynucleotidyltransferase terminal isoforms. A 20-amino acid insertion in the highly conserved carboxyl-terminal region modifies the thermosensitivity but not the catalytic activity.

Terminal deoxynucleotidyltransferase (TdT) catalyzes the addition of nucleotides to 3'-hydroxyl ends of DNA strands in a template-independent manner and has been shown to add N-regions to gene segment junctions during V(D)J recombination. TdT is highly conserved in all vertebrate species, with a second isoform, characterized by a 20-amino acid insertion near the COOH-terminal end, described only in the mouse. The two murine isoforms differ in their subcellular localization, and the long isoform (TdTL) has previously been found to be unable to add N-regions. Using purified protein produced in a high level expression system in Escherichia coli, we were able to carry out detailed catalytic comparisons of these two TdT isoforms. We discovered that TdTL exhibits terminal transferase activity with kinetic parameters similar to those of the conserved TdT isoform (TdTS). We observed, however, that TdTL is inactivated at physiologic temperature but stable at lower temperatures. This thermal sensitivity of TdTL polymerase activity is not correlated with a significant change in the circular dichroism spectrum of the protein. Thus, the 20-amino acid insertion in TdTL does not affect the catalytic activity but modifies the thermosensitivity.

Amino Acid Sequence↗
PubMed2000

Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line.

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.

Animals↗
PubMed2000

Comparison of mouse and rabbit Ei kappa enhancers indicates that different elements within the enhancer may mediate activation of transcription and recombination.

The intronic Ig kappa-light chain enhancer (Eikappa) has been implicated in regulation of transcription and Vkappa-Jkappa recombination at the kappa locus. To identify sequences within the Eikappa enhancer which are involved in control of recombination, we have made use of the finding that the Eikappa element from the rabbit b9 kappa locus is capable of inducing rearrangement, but not transcription of kappa genes in mouse lymphoid cells. We have therefore compared the binding of murine nuclear proteins to the mouse and rabbit Eikappa elements. DNase I footprinting and gel mobility shift assays indicate that only the kappaB, kappaE1, and kappaE2 sites of the rabbit enhancer are able to interact with murine trans-acting factors. Moreover, although the rabbit kappaB site binds murine NF-kappaB p50/p50 and p50/p65 complexes with high affinity, this site is not capable of mediating transcriptional activation of transient transfection reporter constructs in mouse B lineage cells. These results therefore suggest that, in contrast to the maintenance of kappa enhancer transcription which requires all of the Eikappa sites, only the kappaB, kappaE1, and kappaE2 sites may be necessary for the recombinational activity of the enhancer. Furthermore, NF-kappaB-mediated effects on transcription and recombination appear to involve separate downstream activation pathways.

Amino Acid Motifs↗
PubMed2000

Rodent submandibular gland peptide hormones and other biologically active peptides.

The cervical sympathetic trunk-submandibular gland neuroendocrine axis plays an integral role in physiological adaptations and contributes to the maintenance of systemic homeostasis, particularly under the 'stress conditions' seen with tissue damage, inflammation, and aggressive behavior. The variety of polypeptides, whose release from acinar and ductal cells is under sympathetic nervous system control, offers coordinated and progressive levels of endocrine communication. Proteolytic enzymes (e.g. the kallikreins and furin maturases) are involved in the conversion of inactive precursors (e. g. Pro-EGF and SMR1) into biologically active molecules (e.g. EGF, SMR1-pentapeptide), which act on local or distant targets and thereby modulate the homeostatic process.

Animals↗
PubMed2000

Crystallization of the catalytic domain of murine terminal deoxynucleotidyl transferase.

The catalytic domain of murine terminal deoxynucleotidyl transferase (TdT) has been crystallized in the space group P2(1)2(1)2(1), with unit-cell parameters a = 47.1, b = 86.2, c = 111.7 A. The crystals diffract to a resolution of 2.4 A using synchrotron radiation and a full data set has been collected from the native crystals. The enzyme was shown to be active in the crystalline state.

Animals↗
PubMed1999

Methylation of the promoter region may be involved in tissue-specific expression of the mouse terminal deoxynucleotidyl transferase gene.

The terminal deoxynucleotidyl transferase gene (TdT) is expressed in mice only in early B and T lymphoid precursors a few days after birth. Transactivating factors have been shown to contribute to the lymphoid specific expression of TdT, but they do not account entirely for the restriction of its expression to early precursors. Since tissue-specific expression can be modulated by other mechanisms such as DNA methylation and DNA accessibility, we evaluated the methylation pattern of the TdT gene in various expressing and non-expressing tissues and cell lines. Lymphoid and non-lymphoid organs differed significantly in their methylation profiles. In the thymus nearly complete demethylation of a Hha I site in the promoter was associated with high levels of TdT transcription. There was similar, but weaker demethylation of the TdT promoter in bone marrow, possibly due to the presence of a few TdT expressing B cell precursors. The same methylation status was also associated with TdT expression in different B and T cell lines. Kinetic studies of TdT gene demethylation and TdT transcription during thymus development showed that changes in methylation status were also involved in the differential expression of TdT in fetal and adult life. Footprinting experiments revealed the existence of three regions specifically protected by nuclear extracts from TdT -expressing cells. Together, these results suggest that promoter demethylation is involved in the control of TdT expression and implicate new promoter regions in this regulation.

Age Factors↗
PubMed1999

Tubular morphogenesis and mesenchymal interactions affect renin expression and secretion in SIMS mouse submandibular cells.

We have previously immortalized a mouse submandibular gland (SMG) ductal epithelial cell line, SIMS, from pubertal male mice transgenic for the SV40 large T antigen under the control of the adenovirus 5 E1A promoter. Here we demonstrate the role of the extracellular environment in directing not only the morphogenetic behavior of the cells, but also their functional differentiation in terms of renin expression and secretion. First, we measured renin activity of polarized SIMS cells. Low levels of renin are secreted from both the apical and the basolateral domains; the mechanism appears to be direct as no renin was found to be transcytosed across the cell. Second, we studied homotypic and heterotypic mesenchymal cell interactions with SIMS cells. We found that epithelial-mesenchymal coculture in collagen I gels results in branching tubular morphogenesis of SIMS cells and that significant amounts of renin are secreted, probably into the lumen, as the precursor form, prorenin. Third, we investigated the effects of the basement membrane on SIMS cell morphology and function and found that this structure alone is sufficient to allow expression and secretion of both prorenin and active renin. Finally, we established that SIMS cells can express androgen-regulated genes in a transient transfection assay. In addition, in Matrigel cultures androgen receptor expression appears to be induced, suggesting that the SIMS cell line will be useful for further studies on the molecular basis of the observed high-level expression of SMG-specific genes in male mice.

Animals↗
PubMed1999

Extensive junctional diversity in Ig light chain genes from early B cell progenitors of mu MT mice.

Nontemplated (N) nucleotide additions contribute significantly to the junctional diversity of all Ag receptor chains in adult mice except Ig light (L) chains, primarily because terminal deoxynucleotidyl transferase (TdT) expression is turned off at the time of their rearrangement in pre-B cells. However, because some Ig L chain gene rearrangements are detectable earlier during B cell ontogeny when TdT expression is thought to be maximal, we have examined the junctional processing of kappa- and lambda-chain genes of CD45(B220)+CD43+ pro-B cells from mu MT mice. We found that both kappa and lambda coding junctions formed in these B cell precursors were extensively diversified with N-region additions. Together, these findings demonstrate that Ig L chain genes are equally accessible to TdT in pro-B cells as Ig heavy chain genes. Surprisingly, however, the two L chain isotypes differed in the pattern of N addition, which was more prevalent at the lambda-chain locus. We observed the same diversity pattern in pre-B cells from TdT-transgenic mice. These results suggest that some aspects of TdT processing could be influenced by factors intrinsic to the sequence of Ig genes and/or the process of V(D)J recombination itself.

Animals↗
PubMed1998

Extensive junctional diversity of Ig heavy chain rearrangements generated in the progeny of single fetal multipotent hematopoietic cells in the absence of selection.

We analyzed the progeny of individual multipotent hemopoietic cells, derived from the para-aortic splanchopleura, the earliest identified source of lymphocyte precursors in pre-liver mouse embryos. Single precursors were expanded in an in vitro culture system that permits both commitment and differentiation of B cell precursors. We show that from one single multipotent progenitor we could obtain large numbers of B cell precursors that rearrange the Ig heavy chain genes and generate a repertoire as diverse as that observed in adult populations. N region additions are present at V(D)J junctions, showing that terminal deoxynucleotidyl transferase expression has been switched on and is not, consequently, an intrinsic property of adult stem cells. Throughout the culture period, cells show a majority of DJ vs V(D)J rearrangements and a ratio of 2:1 of nonproductive to productive V(D)J rearrangements, which is close to the expected frequency in the absence of selection. In addition, counterselection for D-J rearrangements in reading frame 2 is observed in V(D)J joints, and allelic exclusion was consistently observed. We conclude that of the three events associated with heavy chain rearrangement, two of them, namely allelic exclusion and counterselection of cells in which the D segment is in reading frame 2, are intrinsic to the cell, while selection of productive heavy chain rearrangements is induced in the bone marrow environment.

Animals↗
PubMed1998

High-level expression of murine terminal deoxynucleotidyl transferase in Escherichia coli grown at low temperature and overexpressing argU tRNA.

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.

Animals↗
PubMed1998

Acinar cells are target cells for androgens in mouse submandibular glands.

The variable coding sequence (VCS) multigene family encodes diverse salivary proteins, such as the SMR1 prohormone and the PR-VB1 proline-rich protein in the rat. In situ hybridization was used to study the cell-specific expression of two new mouse VCS genes, Vcs1 and Vcs2. We show that the Vcs1 transcripts, which code for a proline-rich protein, MSG1, are highly abundant in male and female parotid glands, in which they are specifically detected in acinar cells. No expression was seen in the submandibular or sublingual glands. In contrast, Vcs2 transcripts were found only in the acinar cells of the submandibular glands (SMGs) of male mice, in which they are expressed in response to androgens. Expression was found to be heterogeneous within acinar structures. No Vcs2 transcripts were detected in the SMGs of females or castrated males by Northern blot, RNase protection, or in situ hybridization. Androgen administration to females or castrated males induced expression at a level comparable to that of intact males. The Vcs2 gene is the first example of a mouse androgen-regulated gene that is expressed in SMG acinar cells. This result, in addition to our previous observation on SMR1 expression in rats, demonstrates that both acinar cells and granular convoluted tubule (GCT) cells are target cells for androgen action in rodent SMG.

Androgens↗
PubMed1997

The mouse Vcs2 gene is a composite structure which evolved by gene fusion and encodes five distinct salivary mRNA species.

Genes of the VCS (variable coding sequence) family are characterized by an extensive evolutionary divergence in the protein-coding sequence. The VCS family has been characterized by cDNA cloning from submandibular glands in the rat, mouse and humans. At the genomic level, the sequences of two members of this family are known in the rat Rattus norvegicus: the VCSA1 gene, encoding the prohormone-like polypeptide SMR1, and the VCSB1 gene, encoding a salivary Pro-rich polypeptide. No genomic data were available for the VCS genes of other species. To understand the evolution of the VCS gene family better, we have now sequenced 23 kilobases (kb) of the mouse Vcs2 gene. The Vcs2 sequence reveals numerous genomic reorganizations such as an inversion, insertions of short elements and an unusually high number of long interspersed repeated elements (LINEs), which make up 42% of this region. Interestingly, Vcs2 is composed of three different VCS-like regions. The first of these regions contains all the exons necessary to encode the previously described mouse submandibular gland polypeptide MSG2alpha. This region aligns with the entire genomic sequences of rat VCSA1 and VCSB1 genes. The two other regions align with fragments of these rat sequences. The three regions are arrayed in tandem and flanked by LINEs. In particular, the third region also contains exons that were found in mRNA species from the submandibular gland. In total, we have characterized five mRNAs from mouse submandibular glands which have in common their first exon, and are produced by alternative splicing. Vcs2 is thus a single gene that arose by the fusion of three genes (or pseudogenes) of the VCS multigene family.

Alternative Splicing↗
PubMed1997

Constitutive expression of terminal deoxynucleotidyl transferase in transgenic mice is sufficient for N region diversity to occur at any Ig locus throughout B cell differentiation.

N region diversity in Ag receptors is a developmentally regulated process in B and T cells that correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). Absent in fetal and newborn mice, TdT expression is restricted to early T and pro-B cells in adults. To extend the TdT expression pattern throughout B cell ontogenesis, we generated transgenic mice carrying a TdT cDNA under the regulatory elements of the N-myc gene and the IgH enhancer. High expression was observed in secondary lymphoid organs consistent with TdT activity beyond the pre-B cell stage. This suggests that TdT transgene expression is not down-regulated as is the endogenous gene. Unlike normal mice, extensive N region diversity was found in rearranged lambda light chain genes of adult transgenic animals. Therefore, expression of TdT appears sufficient for N region diversity to occur at any Ig locus. More importantly, expression of the transgene takes place during fetal development. As a consequence, the potential fetal B cell repertoire is modified as both rearranged heavy and light chain genes now show N region additions. Constitutive expression of TdT throughout B cell differentiation does not therefore appear deleterious and suggests that TdT is recruited only to participate in the V(D)J recombination process.

Animals↗